Galectin-3 regulates myofibroblast activation and hepatic fibrosis -- Henderson et al. 103 (13): 5060 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Henderson et al. 10.1073/pnas.0511167103.

Supporting Information

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Supporting Figure 6
Supporting Materials and Methods
Supporting Figure 7

Fig. 6. Disruption of the Galectin-3 gene does not affect initial liver injury or inflammatory cell infiltrate after CCL₄ treatment. Mice were treated with a single i.p. injection of CCL₄ or olive oil control, and livers were harvested at days 1, 2, 4, and 7 (n = 6 mice in each group at each time point). (a) Hematoxylin/eosin staining of livers 24 h after vehicle (olive oil) or CCl₄ treatment in WT and Galectin-3^-/- mice. (b) Quantitation of serum alanine aminotransferase (ALT) release 24 h after acute CCL₄-induced liver injury. (c) Macrophage infiltration in WT (filled bars) and Galectin-3^-/- (open bars) livers in control (day 0) and 1, 2, 4, and 7 days post-CCL₄-induced liver injury measured by F4/80 staining and cell counting. (d) Neutrophil infiltration in WT (filled bars) and Galectin-3^-/- (open bars) livers in control (day 0) and 1, 2, 4, and 7 days post-CCL₄-induced liver injury measured by Gr-1 staining and cell counting. (e) FACS characterization of WT and Galectin-3^-/- bone marrow-derived macrophages (BMDMs) matured for 7 days using F4/80 and CD11b. (f) Proinflammatory cytokine profiles of WT and Galectin-3^-/- BMDMs activated with IFN-g/LPS.

Supporting Figure 7

Fig. 7. Galectin-3 regulates myofibroblast activation and hepatic fibrosis despite similar levels of TGF-b expression and signaling. (a) Real-time PCR quantitation of TGF-b expression in whole liver homogenates from control (olive oil vehicle) and chronic (8 weeks) CCL₄-treated WT and Galectin-3^-/- mice (n = 6 mice in each group) (P value was not significant). (b and c) TGF-b activity measured by ELISA in supernatants recovered from mature WT and Galectin-3^-/- bone marrow-derived macrophages (BMDMs) (b) and supernatants (c) recovered from day 4 hepatic stellate cells (HSCs) cultured for 24 h in serum free (SF) media. (d) Smad activation. HSCs (day 4) were quiesced in serum-free media for 24 h and stimulated with TGF-b for 60 min. Lysates were Western blotted for phosphorylated Smad2 (pSmad2) and phosphorylated Smad3 (pSmad3). (e) Western blot analysis of a-SMA expression in HSCs (day 4) quiesced in serum-free (SF) media for 24 h and stimulated with TGF-b (5 ng/ml) for 48 h. (f and g) Real-time PCR quantitation of a-SMA expression (f) and procollagen (I) expression (g) in HSCs cultured in the presence of TGF-b (5 ng/ml) for 48 h. ***, P < 0.0001 compared with WT. (h) Indirect immunofluoresence for a-SMA expression (green) in HSCs (day 4) quiesced in serum-free (SF) media for 24 h and stimulated with TGF-b (5 ng/ml) or TGF-b plus recombinant Galectin-3 (30 mg/ml) for 48 h. (i) Western blot of a-SMA expression. (j) Real-time PCR quantitation of a-SMA expression. (k) Real-time PCR quantitation of procollagen (I) expression. ***, P < 0.0001 compared with WT. (l) Western blot of ERK1/2 and PKC activation in response to PDGF-BB (50 ng/ml) in WT, knockdown with Gal-3 siRNA, and Galectin-3^-/- HSCs.

Supporting Materials and Methods

Human Biopsy Specimens.
Archival human liver samples were obtained from the University of Edinburgh Department of Pathology files. Alanine Aminotransferase (ALT) Determination.
Serum was stored at -80°C until use for determination of ALT levels by an automated enzyme assay (Olympus 20700 analyzer). Immunohistochemistry.
Primary antibodies used: Anti-aSMA clone 1A4 (Sigma), anti-mouse Galectin-3 clone 8942F (Cedarlane Laboratories), anti-human Galectin-3 clone 9C4 (NovoCastra), anti-rat Galectin-3 [a kind gift from F. T. Liu (University of California at Davis)], anti-mouse Gr-1 clone RB6 8C5 (BD Pharmingen), anti-mouse F4/80 clone CI:A3-1 (Serotec). Species appropriate isotype control antibodies were also used for each experiment. Bone Marrow-Derived Macrophage (BMDM) Preparation and Cytokine Measurement.
BMDMs were prepared from WT and Galectin-3^-/- mice as described in ref. 1. Wells were treated with lipopolysaccharide (LPS) (100 ng/ml) and murine IFN-g (IFNg) (100 units/ml) in serum-free media. After a 24-h incubation, supernatants were harvested, and cytokine secretion was determined by a cytometric bead array (BD Pharmingen). TGF-b and TNF-a levels were measured by ELISA (R & D Systems). Preparation of siRNAs.
The sense and antisense strands of mouse Galectin-3 siRNA (where P represents phosphate) were as follows: Gal-3 (seq 1), beginning at nt 502, 5'-GAUGUUGCCUUCCACUUUAdTdT-3' (sense), 5'-PUAAAGUGGAAGGCAACAUCdTdT-3' (antisense); Gal-3 (seq 2), beginning at nt 4, 5'-GCAGACAGCUUUUCGCUUAdTdT-3' (sense), 5'-PUAAGCGAAAAGCUGUCUGCdTdT-3' (antisense); Gal-3 (seq 3), beginning at nt 678, 5'-GGUCAACGAUGCUCACCUAdTdT-3' (sense), 5'-P UAGGUGAGCAUCGUUGACCdTdT-3' (antisense); Gal-3 (seq 4), beginning at nt 190, 5'-GGACAGGCUCCUCCUAGUGdTdT-3' (sense), 5'-P CACUAGGAGGAGCCUGUCCdTdT-3' (antisense). The sense and antisense strands of human siRNA were as follows: Gal-3 (seq 1), beginning at nt 550, 5'– GAAGAAAGACAGUCGGUUUdTdT-3' (sense), 5'-P AAACCGACUGUCUUUCUUCdTdT-3' (antisense); Gal-3 (seq 2), beginning at nt 518, 5'-GCAAUACAAAGCUGGAUAAdTdT-3' (sense), 5'-P UUAUCCAGCUUUGUAUUGCdTdT-3' (antisense); Gal-3 (seq 3), beginning at nt 660, 5'-GUACAAUCAUCGGGUUAAAdTdT-3' (sense), 5'-P UUUAACCCGAUGAUUGUACdTdT-3' (antisense); Gal-3 (seq 4), beginning at nt 658, 5'-CAGUACAAUCAUCGGGUUAdTdT-3' (sense), 5'-P UAACCCGAUGAUUGUACUGdTdT-3' (antisense). Control duplex was siCONTROL nontargeting siRNA no. 2 (Dharmacon Research, Lafayette, CO). Real-time PCR.
Primers and probes were as follows: Mouse procollagen (I), for 5'-TTCACCTACAGCACGCTTGTG-3', rev 5'-GATGACTGTCTTGCCCCAAGTT-3' and probe FAM 5'-ATGGCTGCACGAGTCACA-3' TAMRA. Mouse a-SMA for primer 5'-TCAGCGCCTCCAGTTCCT-3', rev primer 5'-AAAAAAAACCACGAGTAACAAATCAA-3' and probe FAM 5'-TCCAAA TCATTCCTGCCCA-3' TAMRA. Mouse Galectin-3 for primer 5'-TTGAAGCTGACCACTTCAAGGTT-3', rev primer 5'-AGGTTCTTCATCCGATGGTTGT-3' and probe FAM 5'-CGGTCAACGATGCTCACCTACTGCA-3' TAMRA. Mouse TGF-b for primer 5'-CACCGGAGAGCCCTGGATA-3', rev primer 5'-TGTACAGCTGCCGCACACA-3' and probe FAM 5'-CAACTATTGCTTCAGCTCCACAGAGAAGAACTG-3' TAMRA. Human procollagen (I) primers and probe were as follows: for primer 5'-CAAGAGGAAGGCCAAGTCGAGG-3', rev primer 5'-CGTTGTCGCAGACGCAGAT-3' and probe FAM 5'-CCTCAGGTACCATGACCGAGACGTGTGGAAACC-3' TAMRA. 18S rRNA Taqman primer probe mix was purchased from Applied Biosystems.

1. Duffield, J. S., Erwig, L. P., Wei, X., Liew, F. Y., Rees, A. J. & Savill, J. S. (2000) J. Immunol. 164, 2110–2119.